Project Summary The World Health Organization estimates that the global prevalence of diabetes in adults is 9%. Both Type 1 and Type 2 diabetes involve the issue of reduced ?-cell mass; subsequently a cure for diabetes must involve ?-cell replacement. Ideally, a cure would involve inducing regeneration via ?-cell neogenesis from endogenous pancreatic progenitors. For these reasons we are interested in explicating the process of ?-cell neogenesis, i.e., how ? cells are formed from progenitors in the pancreas. Unlike their mammalian counterparts, we have shown that zebrafish readily regenerate their ? cells following cell-specific ablation. Our goal is to identify the mechanisms behind the zebrafish?s capacity for ?-cell neogenesis. Such molecular pathways could then be pharmacologically exploited in humans to induce ?-cell neogenesis. We have recently made two discoveries critical to understanding how zebrafish so easily recover following ?- cell ablation. First, we identified the progenitor source for ?-cell neogenesis?namely a cell type called the centroacinar cell (CAC); second, we discovered that diminished activity of the Sox9b transcription factor leads to significantly accelerated regeneration. From these insights we hypothesized 1) Sox9b acts cell autonomously to maintain progenitor potency in adult CACs; 2) Diminishing Sox9b activity alters the behavior of CACs or their progeny in regeneration; and 3) The identification of downstream genes of SOX9 will elucidate molecular mechanisms that regulate ?-cell differentiation. By testing these hypotheses in three complementary yet independent aims, we expect to discover if the differences in regeneration between sox9b heterozygotes and wildtypes is due either to pre-existing differences in morphology or the behavior of the CACs during regeneration. Furthermore, we will use genomic approaches to identify the direct downstream transcriptional targets of Sox9 homologs because we expect these targets will be the mediators of the sox9b haploinsufficient phenotype. As part of our preliminary data we knocked down SOX9 levels in the human PANC-1 cell line, a surrogate for pancreatic progenitors, and identified affected transcript levels. We have also used chromatin immunoprecipitation and deep sequencing (ChIP-seq) to identify where SOX9 binds in the PANC-1 genome. Putting these results together has allowed us to find direct targets of SOX9 transcriptional activity in PANC-1 cells. We have identified interesting genes downstream of SOX9, such as EpCAM, and identified biological pathways that SOX9 controls, such as cilia function and Notch regulation. Greatly encouraged by our preliminary results we now aim to expand this analysis to find SOX9 targets during development and regeneration. By the end of this proposed work we expect a better understanding of SOX9 function and the discovery of potential therapeutic routes to alleviate ?-cell paucity in humans.